We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for small pox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), systems that model HIV-infection of humans. We use a prime-boost strategy, first immunizing with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We recently conducted a pre-clinical vaccine study in rhesus macaques and investigated cellular immune responses elicited by the vaccine regimen. Of note, while cellular immunity did not influence SIV acquisition, vaccine-induced CD8 T cells not unexpectedly correlated with decreased viremia. This correlation was also observed in male macaques, of particular interest since mucosal immunity and B cell responses were associated with delayed SIV acquisition in females. An on-going pre-clinical study will determine if these protective responses observed in males and females will be reproduced. Gamma-delta T cells, effector cells that are especially important with regard to mucosal immune protection, have also been investigated in these preclinical studies, which have focused in particular on the female reproductive tract, as well as rectal tissue and blood. We have characterized the various cell populations phenotypically and functionally, and shown that gamma-delta T cells in the ectocervix and endocervix, as well as in the circulating population, contribute to control of viremia. These observations, especially regarding the female reproductive tract, are novel and applicable not only to control of HIV/SIV infection, but most likely to mucosal pathogens in general. We have also studied effector cells which mediate non-neutralizing antibody responses which have been shown to contribute to protective efficacy. Natural killer (NK) cells are a key population which can kill target cells directly and also mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC). A comparison of NK cells in uninfected and chronically infected macaques revealed that the natural cytotoxic capacity of the cells was lost with SIV infection, and a greater concentration of IL-15 was necessary to restore this capability. In contrast the ability to mediate Fc effector functions was not impaired. A current study is exploring whether memory NK cells exist in non-human primates (as has been shown in mice) and whether vaccination impacts this cell subset. Finally, T follicular helper cells (Tfh cells) are critical for promoting B cell development and maturation and induction of potent antibody responses. They also provide targets for HIV/SIV infection. A detailed study of these cells in lymph nodes of vaccinated macaques is underway along with concurrent development of memory B cells in germinal centers. A greater understanding of factors that contribute to development of this cell population should enhance the ability to promote more potent antibody responses.